Thermal performance curves (TPCs), which quantify how an ectotherm's body temperature (T b ) affects its performance or fitness, are often used in an attempt to predict organismal responses to climate change. Here, we examine the key -but often biologically unreasonable -assumptions underlying this approach; for example, that physiology and thermal regimes are invariant over ontogeny, space and time, and also that TPCs are independent of previously experienced T b. We show how a critical consideration of these assumptions can lead to biologically useful hypotheses and experimental designs. For example, rather than assuming that TPCs are fixed during ontogeny, one can measure TPCs for each major life stage and incorporate these into stage-specific ecological models to reveal the life stage most likely to be vulnerable to climate change. Our overall goal is to explicitly examine the assumptions underlying the integration of TPCs with T b , to develop a framework within which empiricists can place their work within these limitations, and to facilitate the application of thermal physiology to understanding the biological implications of climate change.

Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virusspecific multiplex PCR primers were developed based on the conserved sequences of all available respiratoryvirus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.

The invasive signal amplification reaction has been previously developed for quantitative detection of nucleic acids and discrimination of single-nucleotide polymorphisms. Here we describe a method that couples two invasive reactions into a serial isothermal homogeneous assay using fluorescence resonance energy transfer detection. The serial version of the assay generates more than 10 7 reporter molecules for each molecule of target DNA in a 4-h reaction; this sensitivity, coupled with the exquisite specificity of the reaction, is sufficient for direct detection of less than 1,000 target molecules with no prior target amplification. Here we present a kinetic analysis of the parameters affecting signal and background generation in the serial invasive signal amplification reaction and describe a simple kinetic model of the assay. We demonstrate the ability of the assay to detect as few as 600 copies of the methylene tetrahydrofolate reductase gene in samples of human genomic DNA. We also demonstrate the ability of the assay to discriminate single base differences in this gene by using 20 ng of human genomic DNA.

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A long-standing hypothesis in evolutionary biology is that polyploid plants have a fitness advantage over diploids in climatically variable or extreme habitats. Here we provide the first empirical evidence that polyploid advantage in these environments is caused by two distinct processes: homeostatic maintenance of reproductive output under elevated abiotic stress, and fixed differences in seed development. In an outdoor climate manipulation experiment using coastal to inland Australian populations of the perennial grass Themeda triandra Forssk., we found that total output of viable seed in drought- and heat-stressed tetraploid plants was over four times higher than in diploids, despite being equal under more favourable growing conditions. Tetraploids also consistently produced heavier seeds with longer hygroscopic awns, traits which increase propagule fitness in extreme environments. These differences add to fitness benefits associated with broader-scale local adaptation of inland T. triandra populations to drought stress. Our study provides evidence that nucleotypic effects of genome size and increased reproductive flexibility can jointly underlie polyploid advantage in plants in stressful environments, and argue that ploidy can be an important criterion for selecting plant populations for use in genetic rescue, restoration and revegetation projects, including in habitats affected by climate change.